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Received: 9 August 2019    Revised: 26 September 2019    Accepted: 14 December 2019

DOI: 10.1111/jfpp.14370

ORIGINAL ARTICLE

Comparison of 11 rice bran stabilization methods by analyzing

lipase activities

Cheng-wei Yu  | Qi-rui Hu  | Hao-wei Wang | Ze-yuan Deng

State Key Laboratory of Food Science

and Technology, Nanchang University, Abstract
Nanchang, China The production of rice bran oil is severely impeded because of the rapid rancidity
Correspondence caused by rice bran lipase. Eleven stabilization treatments including 6 heating treat-
State Key Laboratory of Food Science and ments and 5 nonheating treatments were carried out in this research. Lipase activi-
Technology, Nanchang University, No. 235
Nanjing East Road, Nanchang 330047, ties as well as oil colors, oil compositions, and contents of vitamin E and γ-oryzanol
Jiangxi, China. after different treatments were investigated to evaluate the effect of stabilization
Email: dengzy@ncu.edu.cn
and oil qualities. All the treatments had no significant influence (p > .05) on the oil
Funding information compositions and γ-oryzanol contents. Among six heating treatments, autoclaving
National Natural Science Foundation of
China, Grant/Award Number: 31872890; had the best stabilization effect for 10.73% lipase activity remained and other heat-
State Key Laboratory of Food Science and ing treatments held about 30%–35% lipase activities residual except dry heating
Technology Free Orientation Project,
Grant/Award Number: SKLF-ZZB-201709 (68.88%). Heating treatments except steam heating resulted in the significant de-
creases (p < .05) of oil acid values and significant increases (p < .05) of peroxide val-
ues, meanwhile six heating treatments increased oil colors. Among five nonheating
treatments, ultraviolet irradiation for 18 hr and extreme low temperature (−80°C) for
72 hr were found 57.08% and 58.85% lipase activities residual, respectively. Peroxide
value was significantly increased (p < .05) only by ultrasound. All nonheating treat-
ments had little effect (p > .05) on oil acid values and color. In conclusion, ultraviolet
irradiation might be a potential, convenient, and energy-saving stabilization method
with high oil quality.
Practical applications
Rice bran has a potential for producing about 8 million tons of rice bran oil world-
wide but nearly 90% rice bran was wasted every year. The processing of rice bran is
impeded because of the rancidity caused by lipase, so the stabilization of rice bran is
very important. This research provided a new and time-saving method for evaluating
stabilization efficacy instead of long time storage. Using this new evaluating method,
11 stabilization methods were compared and methods like microwave, extrusion,
and ultraviolet irradiation were considered to be suitable methods for industrial pro-
cessing. In additional, ultraviolet irradiation was found to be a potential, convenient,
and energy-saving stabilization method without influencing oil quality and nutrient
content.

J Food Process Preserv. 2020;00:e14370. wileyonlinelibrary.com/journal/jfpp |

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https://doi.org/10.1111/jfpp.14370
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1 |  I NTRO D U C TI O N & Rattanathanan, 2014). Although these rice bran stabilization
methods have been reported, the utilization rate of rice bran is
Rice is the most important cereal crop and a staple food for more still extremely low; that is, because the major rice processing fac-
than half of the world's population (María et al., 2016; Sharif, Butt, tories in developing countries are lack of stabilization equipment
Anjum, & Khan, 2014). Rice bran, a major by-product of rice mill- or too high energy and equipment cost lead to the low utilization
ing, constitutes 8%–10% of the total weight of rice (Wang, Khir, rate. Therefore, developing a potential, convenient, and energy-
Pan, & Yuan, 2017). It is a rich source of protein (14%–16%), fat saving stabilization method is very vital for the industry of rice
(12%–23%), crude fiber (8%–10%), carbohydrates, vitamins, min- bran processing.
erals, essential unsaturated fatty acids, and phenolics (Wang, Khir, Furthermore, the majority of reported researches about stabi-
et al., 2017). Rice bran is a potential plant material for protein lizing rice bran are long period of storage observation, researchers
production and essential amino acid because of its no allergy and must stabilize a large scale of rice bran and extract the oil for each
low price (Xia et al., 2012). Rice bran hemicelluloses and defatted long experiment. For example, it took Patil 90 days to evaluate the
rice bran have great potential in food industry, especially in the influence of microwave stabilization on rice bran storage (Patil et al.,
development of functional foods such as functional bakery prod- 2016). In additional, Dhingra spent 105 days evaluating the effect
ucts (Gul, Yousuf, Singh, Singh, & Wani, 2015; Hu, Huang, Cao, of rice bran storage and Shin even spent 375  days on the experi-
& Ma, 2009). Furthermore, rice bran oil (47% monounsaturated ment (Dhingra et al., 2012; Shin, Godber, Martin, & Wells, 2010).
fatty acids, 33% polyunsaturated fatty acids, and 20% saturated The experiments like those will expend a great deal of rice bran, lots
fatty acids) (Gul et al., 2015) has been considered to be one of the of manpower, energy, time, and storage space. In addition, apart
most valuable and healthiest edible oil because it contains vitamin from manpower and storage space, the rice bran will be influenced
E (tocopherol and tocotrienol), vitamin K, and γ-oryzanol (Khoei by the storage temperature, moisture, and biological contamina-
& Chekin, 2016). γ-oryzanol is a special nutriment in the rice bran tions like pests, mice, or microorganisms. Considering the rice bran
oil (Gul et al., 2015), which has hypocholesterolemic effect and lipase is the main cause of rancidity, our team put forward a new
helps lowering incidences of oxidative stress-related diseases efficient method to measure the results of rice bran stabilization
such as cancer, cardiovascular disorders, inflammation, aging, and in order to overcome above-mentioned problems. Therefore, the
obesity (Amarasinghe, Kumarasiri, & Gangodavilage, 2009; Khoei aim of this research was to evaluate stabilization efficacy of various
& Chekin, 2016; Patil, Kar, & Mohapatra, 2016; Wang, Wang, & treatments by the alteration of rice bran lipase activities instead of
Wang., 2017). Although the rice bran and rice bran oil are so valu- long time storage and to find suitable stabilization methods for rice
able to develop healthy food, less than 10% crude rice bran oil bran. Oil quality indicators such as acid value, peroxide value, oil
is processed into edible oil mainly due to its high value of free color, oryzanol content, and vitamin E content were all taken into
fatty acid caused by the rice bran lipase (Kaimal et al., 2015; Wang, consideration for the evaluation of stabilization efficacy.
Wang, et al., 2017). After the rice is milled, the rice bran lipase get
in touch with the rice bran oil and then cause the hydrolysis of tri-
glycerides into glycerol and free fatty acids (Brunschwiler, Heine, 2 | E X PE R I M E NTA L S EC TI O N
Kappeler, Conde-Petit, & Nyström, 2013; Patil et al., 2016). Data
showed that there is a potential for producing about 8 million tons 2.1 | Materials
of rice bran oil worldwide but nearly 90% rice bran was wasted
every year (Wang, Wang, et al., 2017). So, the utilization of rice Rice bran (passed through a 70-mess sieve) was obtained from
bran is a valuable issue. Sinograin Jiangxi Branch (Nanchang, China). Rice bran oil was pur-
Chemical refining and physical refining of high-free fatty acid chased from local Rainbow Supermarket (Nanchang, China).
rice bran oil lead to large quantity of wastewater and high energy
input (Prasad, 2010). The fundamental reason of high-free fatty
acid content in rice bran is the rice bran lipase, so inactivating 2.2 | Reagents and standards
rice bran lipase is a commonly used method for rice bran stabi-
lization (Ju & Vali, 2005). Up to now, many methods of stabiliz- α-Tocotrienol and δ-tocopherol standards were purchased from
ing rice bran have been reported, such as chemical stabilization Aladdin (Shanghai, China), and γ-oryzanol was purchased from TCL
or refrigeration (Amarasinghe et al., 2009), parboiling or hydro- (Shanghai, China). Pepsin (swine-origin, enzyme activity: 250  U/mg),
thermal treatment (María et al., 2016; Pradeep, Jayadeep, Guha, papain (enzyme activity: 800 U/mg), and HPLC-grade methyl acetate
& Singh, 2014), extrusion (Kim, Byun, Cheigh, & Kwon, 2010), mi- were obtained from Aladdin (Shanghai, China). HPLC-grade n-hexane
crowave treatment (Nordin, Karim, Ghazali, Adzahan, & Sultan, and methanol were purchased from Merck (Darmstadt, Germany).
2014; Patil et al., 2016), infrared radiations (Wang, Khir, et al., Ethanol, acetic acid, petroleum ether, isooctane, potassium hydroxide,
2017), ohmic heating (Dhingra, Chopra, & Rai, 2012), steam heat- sodium thiosulfate, phenolphthalein, and potassium iodide were pur-
ing (Thanonkaew, Wongyai, Mcclements, & Decker, 2012), and bi- chased from Xilong Scientific (Guangdong, China). Soluble starch was
ological enzyme method like papain or other enzymes (Laokuldilok provided by Tianjin Damao Chemical Reagent (Tianjin, China).
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2.3 | Rice bran composition analysis Dry heating

Fresh rice bran (1.5 kg, divided into three parts) was paved on three
Moisture content was determined by keeping rice bran at 105°C until metal trays (70 × 50 cm) and the thickness of rice bran was about
the sample became constant weight. Protein content was analyzed using 2–3  mm. Then it was heated by a drying oven (DGG-9140, Senxin
Kjeldah method. Oil content was analyzed using Soxhlet extraction. Ash experiment instrument co. LTD, Shanghai, China) at 105°C for 30,
content was analyzed using muffle furnace to heat the rice bran at 550°C 60, and 90 min, respectively.
for 5 hr. Crude fiber content was analyzed using automatic fiber deter-
mination machine (F600, Hanon, Shandong, China). Starch content was Infrared heating
analyzed using amylase and hydrochloric acid to hydrolysis the starch of Fresh rice bran (1.5 kg, divided into three parts) was paved on three
rice bran, then titrated and converted the titration result to the starch metal trays (70 × 50 cm) and the thickness of rice bran was about
content. Dietary fiber content was analyzed using α-amylase, protease, 2–3 mm. Then it was heated by an infrared oven (EC01C, AISHIQI,
and glucosidase to eliminate protein and starch of rice bran and then the Guangdong, China) at 105°C for 30, 60, and 90 min, respectively.
treated sample was kept at 105°C until it became constant weight.

2.4.2 | Nonheating treatments
2.4 | Different treatments on the fresh rice bran
Low-temperature treatment
The treatment methods on the fresh rice bran could be classified Fresh rice bran (1.5  kg, divided into three parts) was put in three
into two sorts: heating treatments including microwave, steam heat- plastic zip-lock bags and stored at 4°C fridge (BCD-642WDVMU1,
ing, dry heating, infrared heating, autoclaving, and extrusion; and Haier, Qingdao, China), −18°C fridge (BCD-642WDVMU1, Haier,
nonheating treatments such as enzymatic inactivation (pepsin and Qingdao, China), −80°C fridge (DW-86L828W, Haier, Qingdao,
papain) and low-temperature, ultraviolet irradiation, and ultrasound China) for 72 hr, respectively.
treatment in this research.
Control group: Fresh rice bran without any treatments. Ultraviolet irradiation
Fresh rice bran (1.5  kg, divided into three parts) was paved on
three metal trays (70 × 50 cm) and the thickness of rice bran was
2.4.1 | Heating treatments about 2–3 mm. Then it was put on a bacteria-free operating desk
(JB-CJ-1500FX, Jiabao purification engineering equipment co.
Microwave LTD, Suzhou, China) and irradiated by ultraviolet irradiation for
Fresh rice bran (1.5  kg, divided into three parts) was put in three 6, 12, and 18  hr, respectively. The ultraviolet wavelength was
2,000 ml beakers and then 700 w microwaved (G70F20CN3L-C2S2, 254 nm and the distance between ultraviolet lamp and metal trays
Galanz, Guangdong, China) for 2, 4, and 6 min, respectively. was 45 cm.

Autoclaving Ultrasound
Fresh rice bran (0.5 kg) was put in a 2,000 ml beaker and the beaker Fresh rice bran (1.5  kg, divided into three parts) was put in three
was sealed by two layers of gauze. Then it was treated by an au- 3,000 ml beakers and 2,000 ml distilled water was added to it. The
toclave sterilizer (LDZX-50KBS, Shenan medical equipment factory, beaker was put in an ultrasound cleaner (GA-300SP, Shangjia bio-
Shanghai, China) at 121°C for 20 min. logical technology co. LTD, Wuxi, China) and treated at 28 kHz and
300 w for 30, 60, and 90 min, respectively. After ultrasound treat-
Extrusion ment, the mixture was centrifuged at 4,200 rpm for 5 min and ob-
Fresh rice bran (1  kg, divided into two parts) was added into the tained the sediment (treated rice bran).
extrusion equipment (SLG65-D, Dayi extrusion machinery co. LTD,
Jinan, China). The extrusion equipment was divided into four parts Enzyme treatment
for 60°C, 65°C, 115°C, and 120°C. The screw speeds were 400 and Ten grams of enzyme (pepsin, papain) were dissolved in 2 L distilled
500 rpm, respectively. water to obtain enzyme solution (5 g/L). Rice bran (1.5 kg, divided
into three parts) was added in three 3,000 ml beakers and mixed
Steam heating with 2,000 ml enzyme solution, and then continuously stirred by
Water boiled at 100°C by induction cooker (21HEC05, Joyoung, an electronic blender (JJ-1, Chengdong xinrui instrument factory,
Shandong, China) was used to steam fresh rice bran (1.5 kg, divided Jintan, China) for 30, 60, and 90min, respectively. After enzymatic
into three parts and wrapped by two layers of gauze) for 20, 40, and treatment, the mixture was centrifuged at 4,200 rpm for 5 min and
60 min, respectively. the sediment was obtained (treated rice bran).
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2.5 | Rice bran oil extraction P=

1,000 × V × C
2m

Treated rice bran were mixed with petroleum (1:7, w/v), and then con-
tinuously stirred by an electronic blender (JJ-1, Chengdong xinrui in- where P: Peroxide value (mmol/kg).
strument factory, Jintan, China) for 2 hr. After extraction, the mixture V: The amount of sodium thiosulfate (ml).
was centrifuged at 4,200 rpm for 5 min. The supernatant was evapo- C: The concentration of sodium thiosulfate (mol/L).
rated at 40°C using a rotary evaporator (Eyela N-100, Tokyo, Japan) to m: The mass of rice bran oil (g).
obtain rice bran oil sample. Samples were stored at 4°C until analysis.

2.9 | The extraction of rice bran lipase

2.6 | Rice bran oil color analysis
Treated rice bran was mixed with phosphate-buffered solution
Analyses of oil color were carried out using a standard colorimetric (1:7, w/v), and then continuously stirred by an electronic blender
method according to the Chinese Standard (GB/T 22460-2008/ISO (JJ-1, Chengdong xinrui instrument factory, Jintan, China) for 2  hr.
15305:1998). Automatic lovibond chromometer (PLV-300, Perkone After extraction, the mixture was centrifuged at 4,200 rpm for 5 min
Scientific, Hangzhou, China) was used to measure the color of rice and the supernatant was obtained. The supernatant was subsided by
bran oil. About 5 ml rice bran oil was added in the cuvette and the ammonium sulfate and then dialyzed for 36 hr to obtain crude pro-
machine would show the results automatically. tein. The protein solution was freeze dried by lyophilizer (LGH-1A,
Yataikelong, Beijing, China) to obtain crude rice bran lipase powder.

2.7 | Acid value analysis

2.10 | The activity of rice bran lipase
Analyses of acid value were carried out using a standard titration
method according to the official methods described in Method for Activity of rice bran lipase: The amount of 1 μmol fatty acid released
Analysis of Hygienic Standard of Edible Oils of the People's Republic per minute by 1g lipase was defined as a unit of lipase activity (U).
of China (GB 5009.229-2016). Briefly, 4  g of rice bran oil was dis- One gram rice bran lipase powder was dissolved in 5 ml distilled
solved in 50 ml 95% ethanol. Phenolphthalein was used as indicator. water to make a lipase solution. One milliliter lipase solution was
Potassium hydroxide (0.1 mol/L) was used to titrate the solution until added to 4 g rice bran oil for a reaction at 35°C. After the reaction,
it became red and lasted for 30 s. 50 ml 95% ethanol and 0.5 ml phenolphthalein were added to the
The acid value was calculated as: solution. Potassium hydroxide (0.1  mol/L) was used to titrate the
solution until it became red, which lasted for 30 s.
V × C × 56.11 The activity of rice bran lipase was calculated as:
X=
m
V × C × 1,000
X=
where X: Acid value (KOH mg/g). t×m
V: The amount of potassium hydroxide (ml).
C: The concentration of potassium hydroxide (mol/L). where X: The activity of rice bran lipase (U).
56.11: The molecular weight of potassium hydroxide. V: The amount of potassium hydroxide (ml).
m: The mass of rice bran oil (g). C: The concentration of potassium hydroxide (mol/L).
t: Reaction time (min).
m: The mass of rice bran lipase (g).
2.8 | Peroxide value analysis

Analyses of peroxide value were carried out using a standard titration 2.11 | GC analysis of oil composition
method according to the official methods described in Method for
Analysis of Hygienic Standard of Edible Oils of the People's Republic Gas chromatography (6890N, Agilent Technologies, USA) was per-
of China (GB/T 5538-2005/ISO 3960:2001). Fifty milliliters acetic formed with flame ionization detection and a CP-Sil88 column
acid–isooctane (volume ratio: 3:2) were added to dissolve 3 g rice (CP7489, 100  m  ×  0.25  mm  ×  0.2  μm, Chrompack; Agilent, USA)
bran oil. Then 0.5 ml saturated potassium iodide solution was added under the following conditions: injector temperature was 250°C and
to it and quickly covered for 1 min reaction. Next sodium thiosulfate operated in the splitless mode. Nitrogen was used as a carrier gas;
(0.002 mol/L) was used to titrate the solution. When the solution be- hydrogen and air were used as burning gas, with a flow velocity of
came pale yellow, 0.5 ml starch indicator was added and then the solu- 1.8 ml/min. The temperature of the flame ionization detector (FID)
tion was titrated until it became white. was 250°C; the column pressure was 24.52  psi. The initial column
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temperature was 45°C and held for 5 min, then increased to 175°C Heating treatments are the most widely used method of rice
at a rate of 13°C/min and held for 27 min, and finally programmed bran stabilization in the industrial production. Many researchers
at 4°C/min to 215°C and held for 35  min (Deng et al., 2018). The have pointed out that lipase was unstable and easy to be inacti-
fatty acids were identified by comparing their retention times with a vated at high temperatures (Sharma, Sharma, Pathania, & Handa,
standard FAME mixture (#463). 2017; Ulker & Karaoglu, 2012), which was due to protein denatur-
ation (Sharma et al., 2017), a basic theory of rice bran stabilization.
Dry heating has turned out to be an effective way to stabilize rice
2.12 | HPLC analysis of tocopherols and oryzanol bran by Kim, Chung, and Lim (2014). Among the heating treat inac-
tivation, dry heating for 90 min treatment had the most inefficient
Approximately 1 g of oil sample was put into a volumetric flask and stabilization result (68.88% relative activity remained). The similar
made up to 10 ml with HPLC-grade hexane. After filtering through temperature and heating time for steam heating and infrared heating
a 0.22 µm filter, 3 µl of sample was directly injected into an Agilent had better stabilization results, remained 35.96% and 35.00% lipase
1100 series HPLC system equipped with a diode array detector activity, respectively. Amarasinghe reported a similar conclusion
(DAD). The DAD wavelength was set at 295  nm for tocopherols that the free fatty acid increased from 2.8% to 9.0% for dry heating
and 326 nm for oryzanol. The column was a Hypersil ODS2 (5 µm, and 2.8% to 6.1% for steaming, which proved steaming had a better
4.6  ×  150  mm). The mobile phase was methanol/water (93:7, v/v), stabilization effect than dry heating (Amarasinghe et al., 2009). A
and the flow rate was 1.0 ml/min. The corresponding standards slight increase (6.1%–6.7%) by infrared heating for 6 months of stor-
(α-tocotrienol, δ-tocopherol, and γ-oryzanol) for identification and age was proved by Maria (Irakli, Kleisiaris, Mygdalia, & Katsantonis,
quantification were prepared in hexane solution. The coefficients of 2018). Microwave and extrusion had similar inactivation effects
variation were .9997, .9998, and .9998, respectively. (about 30% lipase activity residual) in contrast to steam heating
and infrared heating but much shorter processing time, especially
microwave. Microwave is efficient, effective, economical, and has
2.13 | Statistical analysis a little effect on the nutriments of rice bran (Nordin et al., 2014).
Sharmila's research proved that free fatty acid and peroxide value of
All experiments were performed in triplicate, and data were ex- microwaved rice bran were both significant lower (p < .05) than un-
pressed as mean value ± standard deviation. All statistical analy- treated rice bran and parboiled rice bran, which indicated the lipase
ses were performed using SPSS 20.0. One-way ANOVA analysis and peroxidase were inactivated effectively. (Sharma et al., 2017)
with Duncan's test was used to compare the differences between Extrusion is a widely used method for the stabilization of rice bran.
mean values. A level of probability at p < .05 was set as statistically Shin found that free fatty acid of extruded rice bran (140°C) was
significant. 20 times lower than raw bran after 350 days storage. The stabiliza-
tion effect became better as the extrusion temperature increased
from 110°C to 140°C (Shin et al., 2010). Interestingly, autoclaving
3 | R E S U LT S A N D D I S CU S S I O N S held the best inactivation effect for 10.73% relative activity re-
sidual not only in heating treatment inactivation but also in all the
3.1 | Rice bran composition treatments studied in our research. This phenomenon might due to
the combination of high temperature and high pressure. Kim have
The main compositions of rice bran were moisture (9.39 ± 0.09%), also proved that autoclaving had better inactivation effect than dry
fat and oil (20.00 ± 0.30%), protein (15.45 ± 0.23%), crude fiber heating and microwave heating for 24 weeks storage at room
(5.32 ± 0.14%), ash (7.63 ± 0.16%), starch (11.80 ± 0.17%), and solu- temperature (Kim et al., 2014).
ble dietary fiber (30.41 ± 0.40%). According to the quality classifica- By comparing the results and characteristics of these heating
tion of rice bran (Chinese Standard GB 10371-1989), the rice bran treatments, not all of them are suitable for large-scale industrial
used in this research was the first grade (the best quality) in which processing. Autoclaving is the most effective way to inactivate the
the protein content is over 13%, crude fiber content less than 6%, lipase of rice bran, but autoclaving needs autoclave sterilizer which
and ash content less than 8%. requires high energy and only small scale make it hardly used in in-
dustry. Extrusion is widely used in rice bran stabilization because its
convenience and high efficiency. Another advantage is the structural
3.2 | Effects of different treatments on the damage of rice bran during the processing, which leads to higher oil
lipase activities extraction rate. Microwave had a similar stabilization efficacy in
this research. Compared with extrusion, its processing time is much
The results of different treatments on the lipase activities are shown shorter than extrusion. So the microwave stabilization in industry
in Table 1. It is important to note that autoclaving and extrusion is worth further development. Steam heating and infrared heating
are both the combinations of high-temperature and high-pressure can also be used for large-scale stabilization in industry. But com-
treatment. pared with the convenience and high efficiency of microwave and
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TA B L E 1   Effect of different treatments on the lipase activities

Relative
Treatment Relative activity (%) Treatment Relative activity (%) Treatment Relative activity (%) Treatment activity (%)

Control 100.00 ± 0.00a Control 100.00 ± 0.00a Control 100.00 ± 0.00a Control 100.00 ± 0.00a

b b b
Microwave 700 w 2 min 70.14 ± 1.03 Autoclaving 121°C 10.73 ± 1.54 Extrusion 400 rpm 39.27 ± 1.62 Steam heating 100°C 54.92 ± 2.00 b
20 min 20 min
Microwave 700 w 4 min 45.59 ± 1.46c     Extrusion 500 rpm 31.98 ± 0.81c Steam heating 100°C 37.23 ± 2.49c
40 min
Microwave 700 w 6 min 33.48 ± 0.83d         Steam heating 100°C 35.96 ± 2.96c
60 min
Control 100.00 ± 0.00a Control 100.00 ± 0.00a Control 100.00 ± 0.00a Control 100.00 ± 0.00a
Dry heating 105°C 30 min 67.19 ± 2.43b Infrared heating 63.50 ± 1.17b 4°C 72 hr 99.32 ± 1.16a Ultraviolet irradiation 76.40 ± 3.08b
105°C 30 min 6 hr
Dry heating 105°C 60 min 64.80 ± 0.60 b Infrared heating 53.25 ± 0.97c −18°C 72 hr 97.13 ± 1.33a Ultraviolet irradiation 64.18 ± 0.86c
105°C 60 min 12 hr
Dry heating 105°C 90 min 68.88 ± 2.87b Infrared heating 35.00 ± 1.88d −80°C 72 hr 58.85 ± 1.55b Ultraviolet irradiation 57.08 ± 0.42d
105°C 90 min 18 hr
Control 100.00 ± 0.00a Control 100.00 ± 0.00a Control 100.00 ± 0.00a    
a b a
Ultrasound 28 KHz 300 w 100.38 ± 0.88 Pepsin 30 min 98.29 ± 0.00 Papain 30 min 101.36 ± 1.86    
30 min
Ultrasound 28 KHz 300 w 99.00 ± 1.11a Pepsin 60 min 98.43 ± 0.00 b Papain 60 min 98.38 ± 3.95a    
60 min
Ultrasound 28 KHz 300 w 96.62 ± 1.02b Pepsin 90 min 95.93 ± 0.03b Papain 90 min 80.48 ± 3.27b    
90 min

Note: Means with different superscript letter within the same column for each parameter are significantly different (p < .05).
YU et al.
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extrusion, these two methods cost more time and more energy. So quaternary structure, then lose the catalytic activity. Although the
steam heating and infrared heating are not wise choice in compari- residual lipase activity after ultraviolet irradiation was higher than
son with microwave and extrusion. Dry heating may not be used for heating treatment methods, convenience, low energy cost, little in-
stabilization because of its low efficacy. fluence on the acid value, peroxide value, γ-oryzanol content, and
As for the nonheating treatment, pepsin had almost no inac- tocopherol content are its advantage (results are shown later). In
tivation effect on the lipase, even 95.93% lipase activity residual addition, better stabilization effect can be obtained by optimizing
after 90 min treatment. In comparison with pepsin, papain held a the wavelength, thickness of rice bran, processing time, and other
better inactivation effect of 80.48% remained after 90 min treat- conditions. Therefore, ultraviolet irradiation can be used as a stabi-
ment. Protease like pepsin and papain were reported to be used lization method or pretreatment before other stabilization methods
in rice bran stabilization (Laokuldilok & Rattanathanan, 2014). But like microwave and extrusion. Further researches about improving
owing to the high price of protease and less efficacy, using prote- ultraviolet irradiation stabilization efficacy are needed.
ase for rice bran stabilization is not a wise choice. Low-temperature In summary, measuring the alteration of rice bran lipase activi-
treatment, sometimes called refrigeration in other researches, was ties was a convenient and practicable method to evaluate the stabi-
found to be a suitable way to stabilize rice bran (Kim et al., 2014), lization efficacy of various treatments. The results in this research
mainly due to lipase activity was inhibited at low temperature. No were in consistent of other people's previous researches. Extrusion
significant decrease (p  >  .05) of lipase activity was found at 4°C and microwave are suitable to be used in large-scale and short-time
and −18°C for 72 hr treatment in our research. But for −80°C, the industrial processing. Ultraviolet irradiation was found to be a po-
relative activity was 58.85% remained after 72 hr refrigeration. tential, convenient and energy-saving stabilization method.
The decrease of lipase activity might be due to the protein dena-
turation at ultralow temperature. Low-temperature treatment is
suitable for small-scale storage but not for large scale because it is 3.3 | Effects of different treatments on the
not energy intensive and economical. As for ultrasound treatment, oil colors
little inactivation effect was discovered, which meant it might
not be an effective stabilization method. Surprisingly, ultraviolet The results of oil color were described by the values of four colors
irradiation had a capacity of stabilizing rice bran. The remained (red, yellow, blue, and gray). In Chinese Standard, color of liquid
activities were 76.40% for 6 hr, 64.18% for 12 hr, and 57.08% for oil sample is compared with the standard glasses in the same light
18  hr. Joseph indicated that beta-lactoglobulin had a change in source. Red and yellow are mainly used, blue and neutral color (gray)
protein folding after being irradiated at 295 nm for 24 hr (Kehoe, are used unless it is necessary to adjust the color. The darker oil is
Remondetto, Subirade, Morris, & Brodkorb, 2008). Similarly, corresponding to the higher values, for example, the color of oil A
Kristo used fluorescence spectra and surface hydrophobicity mea- (red: 2.0, yellow: 10.0, blue: 0.2, gray: 0.1) is darker than B (red: 1.6,
surements to prove the changes in the tertiary structure of protein yellow: 9.0, blue: 0.1, gray: 0.0).
with ultraviolet irradiation exposure (Kristo, Hazizaj, & Corredig, As shown in Table 2, the yellow value and red value increased
2012). During ultraviolet irradiation, rice bran lipase might be significantly (p < .05) after dry heating, steam heating, microwave, ex-
denatured and result in the changes of tertiary and quaternary trusion, autoclaving, and infrared heating. These results showed that
structures, then lost the catalytic activity. But there is almost no the color of rice bran oil became darker after these treatments. These
report about stabilizing rice bran using ultraviolet irradiation, it is methods had in common was heating treatment. This phenomenon
still needs further research. If ultraviolet irradiation can be used to might be due to the formation of some products from the Maillard re-
stabilize rice bran, it can be a large-scale, convenient, and low-cost action caused by the heating treatment (Kim et al., 2014). Nonheating
stabilization method. treatments could also influenced the oil color but not so obvious as
By comparing the results and characteristics of these nonheating heating treatments. And pepsin and papain had no significant influ-
treatments, only refrigeration and ultraviolet irradiation are suitable ence on oil color because of the highly specific catalytic activities of
for rice bran stabilization. But refrigeration requires large icehouse enzymes (Nam & Palsson, 2012). Ultraviolet had little influence on
and continuous refrigeration, no doubt it is energy-intensive and un- oil color (red and yellow were 0.80 and 8.17, respectively, after irra-
economic. In addition, low temperature (except −80°C can inacti- diation for 18 hr). Heating treatments make the color of rice bran oil
vate the lipase) just inhibits the lipase activity but cannot inactivate increased, which results in the necessity of decoloring in oil refining.
the lipase. Once the rice bran be taken out from the icehouse, the
free fatty acid content will increase rapidly again. So refrigeration is
an inhibition method just for temporary and small-scale treatment. 3.4 | Effects of different treatments on the acid
Ultraviolet irradiation was found to be a potential method for sta- values and peroxide values
bilization. Ultraviolet irradiation can change the tertiary structure
of protein and cause protein denaturation, which is the theory of As shown in Table 3, significant decreases (p < .05) on acid val-
ultraviolet sterilization. During ultraviolet irradiation, rice bran ues were found in the treatments of microwave, autoclaving,
lipase may be denatured and result in the changes of tertiary and steam heating, low temperature, extrusion, and infrared heating.
 |

TA B L E 2   Effect of different treatments on the oil colors

8 of 14      

Treatment Red Yellow Blue Gray Treatment Red Yellow Blue Gray
a A ① 1 a A ①
Control 1.00 ± 0.00 8.10 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Control 1.00 ± 0.00 8.10 ± 0.00 0.00 ± 0.00 0.00 ± 0.001
ab B ① 2 a B ①
Microwave 700 w 2 min 1.03 ± 0.06 8.83 ± 0.06 0.00 ± 0.00 −0.10 ± 0.00 Autoclaving 121°C 20 min 1.17 ± 0.12 12.87 ± 0.65 0.00 ± 0.00 −0.10 ± 0.002
Microwave 700 w 4 min 1.20 ± 0.00 c 9.47 ± 0.21C 0.00 ± 0.00 ① −0.10 ± 0.002          
Microwave 700 w 6 min 1.10 ± 0.00 b 11.93 ± 0.21D 0.00 ± 0.00 ① −0.10 ± 0.002          
a A ① 1 a A ①
Control 0.85 ± 0.06 8.40 ± 0.06 0.00 ± 0.00 −0.10 ± 0.00 Control 0.90 ± 0.00 8.70 ± 0.00 0.00 ± 0.00 0.00 ± 0.001
Extrusion 400 rpm 1.25 ± 0.06b 12.96 ± 0.15B 0.00 ± 0.00 ① −0.10 ± 0.001 Steam heating 100°C 20 min 1.17 ± 0.06b 12.90 ± 0.20 B 0.00 ± 0.00 ① −0.10 ± 0.002
b B ① 1 b B ①
Extrusion 500 rpm 1.30 ± 0.12 13.07 ± 0.12 0.00 ± 0.00 −0.10 ± 0.00 Steam heating 100°C 40 min 1.17 ± 0.06 13.17 ± 0.35 0.00 ± 0.00 −0.10 ± 0.002
          Steam heating 100°C 60 min 1.10 ± 0.00 b 14.43 ± 0.06C 0.00 ± 0.00 ① −0.10 ± 0.002
a A ① 1 a A ①
Control 0.80 ± 0.00 8.67 ± 0.06 0.00 ± 0.00 0.00 ± 0.00 Control 0.90 ± 0.00 8.30 ± 0.00 0.00 ± 0.00 0.00 ± 0.001
Dry heating 105°C 0.90 ± 0.00 b 9.57 ± 0.12B 0.00 ± 0.00 ① 0.00 ± 0.001 Infrared heating 105°C 0.87 ± 0.06a 8.97 ± 0.06B 0.00 ± 0.00 ① 0.00 ± 0.001
30 min 30 min
Dry heating 105°C 1.00 ± 0.00 c 11.87 ± 0.29C 0.00 ± 0.00 ① 0.00 ± 0.001 Infrared heating 105°C 0.90 ± 0.00a 9.73 ± 0.06C 0.00 ± 0.00 ① 0.00 ± 0.001
60 min 60 min
Dry heating 105°C 1.07 ± 0.06c 14.13 ± 0.12D 0.00 ± 0.00 ① 0.00 ± 0.001 Infrared heating 105°C 1.00 ± 0.00 b 10.63 ± 0.15D 0.00 ± 0.00 ① 0.00 ± 0.001
90 min 90 min
Control 0.80 ± 0.00a 8.67 ± 0.06B 0.00 ± 0.00 ① 0.00 ± 0.001 Control 0.77 ± 0.06ab 8.13 ± 0.06A 0.00 ± 0.00 ① 0.00 ± 0.001
d c ① 1 a B ①
4°C 72 hr 1.00 ± 0.00 9.30 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Ultraviolet irradiation 6 hr 0.70 ± 0.00 8.50 ± 0.00 0.00 ± 0.00 0.00 ± 0.001
−18°C 72 hr 0.90 ± 0.00 b 8.70 ± 0.00 B 0.00 ± 0.00 ① 0.00 ± 0.001 Ultraviolet irradiation 12 hr 0.80 ± 0.00 b 8.54 ± 0.06B 0.00 ± 0.00 ① 0.00 ± 0.001
ab A ① 1 b A ①
−80°C 72 hr 0.83 ± 0.06 8.47 ± 0.06 0.00 ± 0.00 0.00 ± 0.00 Ultraviolet irradiation 18 hr 0.80 ± 0.00 8.17 ± 0.06 0.00 ± 0.00 0.00 ± 0.001
Control 0.77 ± 0.06a 8.13 ± 0.06A 0.00 ± 0.00 ① 0.00 ± 0.001 Control 0.83 ± 0.06a 8.17 ± 0.15A 0.00 ± 0.00 ① 0.00 ± 0.001
Ultrasound 28 KHz 0.93 ± 0.06b 8.80 ± 0.00 C 0.00 ± 0.00 ① 0.00 ± 0.001 Pepsin 30 min 0.90 ± 0.00a 8.40 ± 0.00A 0.00 ± 0.00 ① 0.00 ± 0.001
300 w 30 min
Ultrasound 28 KHz 0.73 ± 0.06a 8.07 ± 0.06A 0.00 ± 0.00 ① 0.00 ± 0.001 Papain 60 min 0.89 ± 0.02a 8.39 ± 0.03A 0.00 ± 0.00 ① 0.00 ± 0.001
300 w 60 min
Ultrasound 28 KHz 0.83 ± 0.06ab 8.47 ± 0.06B 0.00 ± 0.00 ① 0.00 ± 0.001 Papain 90 min 0.90 ± 0.00a 8.38 ± 0.04A 0.00 ± 0.00 ① 0.00 ± 0.001
300 w 90 min
Control 0.83 ± 0.06a 8.17 ± 0.15A 0.00 ± 0.00 ① 0.00 ± 0.001          
a A ①
Papain 30 min 0.89 ± 0.00 8.35 ± 0.06 0.00 ± 0.00 0.00 ± 0.001          
a A ① 1
Papain 60 min 0.88 ± 0.06 8.32 ± 0.00 0.00 ± 0.00 0.00 ± 0.00          
Papain 90 min 0.90 ± 0.00a 8.33 ± 0.06A 0.00 ± 0.00 ① 0.00 ± 0.001          

Notes: Means with different superscript letter or number within the same column for each parameter are significantly different (p < .05).
YU et al.
YU et al. |
      9 of 14

TA B L E 3   Effect of different treatments on the acid values of rice bran oil

Acid value Acid value Acid value

Treatment (KOH mg/g) Treatment (KOH mg/g) Treatment (KOH mg/g)

Control 68.79 ± 0.23b Control 68.79 ± 0.23b Control 50.17 ± 0.23c

Microwave 700 w 2 min 68.73 ± 0.11b Autoclaving 121°C 20 min 60.72 ± 0.12a Extrusion 400 rpm 40.61 ± 0.22b
ab
Microwave 700 w 4 min 66.32 ± 1.84     Extrusion 500 rpm 39.57 ± 0.16a
Microwave 700 w 6 min 64.58 ± 0.27a        
Control 41.60 ± 0.43a Control 44.94 ± 0.11b Control 46.28 ± 0.02b
Steam heating 100°C 40.93 ± 0.81a Dry heating 105°C 30 min 44.11 ± 0.06a Infrared heating 105°C 30 min 46.79 ± 0.28b
20 min
Steam heating 100°C 39.92 ± 2.24a Dry heating 105°C 60 min 45.36 ± 0.16b Infrared heating 105°C 60 min 46.41 ± 0.03b
40 min
Steam heating 100°C 39.41 ± 0.24a Dry heating 105°C 90 min 44.01 ± 0.24a Infrared heating 105°C 90 min 44.98 ± 0.06a
60 min
Control 44.94 ± 0.11d Control 50.27 ± 0.65ab Control 50.27 ± 0.65a
4°C 72 hr 41.69 ± 0.09c Ultraviolet irradiation 6 hr 53.07 ± 0.39bc Ultrasound 28 KHz 300 w 30 min 50.42 ± 0.05a
b c
−18°C 72 hr 40.66 ± 0.22 Ultraviolet irradiation 12 hr 54.62 ± 0.42 Ultrasound 28 KHz 300 w 60 min 50.42 ± 0.13a
−80°C 72 hr 39.74 ± 0.31a Ultraviolet irradiation 18 hr 49.14 ± 1.52a Ultrasound 28 KHz 300 w 90 min 53.64 ± 1.09a
a a
Control 55.04 ± 0.35 Control 55.04 ± 0.35    
a a
Pepsin 30 min 54.81 ± 0.80 Papain 30 min 55.11 ± 1.17    
Pepsin 60 min 54.75 ± 0.27a Papain 60 min 55.26 ± 0. 21a    
a a
Pepsin 90 min 54.87 ± 0.18 Papain 90 min 55.08 ± 0.08    

Notes: Means with different superscript letter within the same column for each parameter are significantly different (p < .05).

Thanonkaew found that the stabilization methods like steaming and could promote the production of free radicals as well as the de-
microwave could lead to lower acid value than unstabilized rice bran composition and polymerization of hydroperoxide (Belitz & Grosch,
(Thanonkaew et al., 2012). Compared with the control group, the 1999). This might be a reasonable explanation for the increased per-
acid values of low-temperature treatment were significantly lower oxide values of heating methods like microwave, autoclaving, dry
after 72 hr treatment because the activity of lipase was inhibited at heating, infrared heating, and extrusion (400 rpm). Kreps reported
the low temperature (Sharma et al., 2017; Ulker & Karaoglu, 2012). that microwaving heating could increase the peroxide value of sun-
Similar finds were reported by Amarasinghe that the free fatty acid flower oil and corn oil (Kreps, Vrbiková, Schmidt, Sekretár, & Híreš,
for unstabilized, refrigerated, and steamed rice bran were 3.12%, 2015). In addition, Javidipour indicated that peroxide values of ha-
2.80%, and 2.80%, respectively (Amarasinghe et al., 2009). Riaz also zelnut oil, soybean oil, and sunflower oil for 3 min microwave and
drew the conclusion that the free fatty acid of extruder (2.72%) was 6 min microwave were higher than nonmicrowaved group's perox-
lower than raw material (5.20%) (Riaz, Asif, Plattner, & Rokey, 2010). ide value, and then decreased when the microwave time prolonged
The other treatments like dry heating, steam heating, ultraviolet ir- to 9 min (Javidipour, Erinc, Basturk, & Tekin, 2016). The 90 min dry
radiation, ultrasound, and enzymatic treatments had no influence on heating at 105°C had 2 times of peroxide value than control group
acid values. and the residual lipase activity was still 68.88%. So the dry heating
The effects of different treatments on peroxide values are was not a good method to stabilize rice bran. Interestingly, steam
shown in Table 4. Peroxide value is a main index to evaluate the de- heating (100°C) and extrusion (500 rpm) were found no significant
gree of oil oxidation. The peroxide value increases with the oxidation increases (p > .05) of peroxide values. This result indicated that the
of oil, and when the oil is deeply oxidized, the decomposition rate of steam heating (100°C) and extrusion (500 rpm) might be two non-
hydroperoxide exceeds the generation rate of hydroperoxide, the oxidation methods for rice bran stabilization.
peroxide value decreases (Belitz & Grosch, 1999). Most of the non-
heating treatments such as ultraviolet irradiation, refrigeration, and
enzyme treatments had no significant influence (p > .05) on perox- 3.5 | Effects of different treatments on the fatty
ide values except ultrasound; on the contrary, heating treatments acid compositions
had a significant increase (p < .05) on peroxide values more or less
except steam heating. The result showed that ultrasound might As shown in the Table 5, six main fatty acids of rice bran oil were
have an activation of oil oxidation reaction. The oxidation rate in- analyzed. The amount of unsaturated fatty acids (oleic acid, linoleic
creased with the increasing temperature because high temperature acid, linolenic acid, and arachidic acid) consisted over 80% of the
 |
10 of 14       YU et al.

TA B L E 4   Effect of different treatments on the peroxide values of rice bran oil

Peroxide value Peroxide Peroxide value

Treatment (mmol/kg) Treatment value (mmol/kg) Treatment (mmol/kg)

Control 13.96 ± 0.03a Control 13.96 ± 0.03a Control 8.36 ± 0.45a

Microwave 700 w 2 min 20.38 ± 0.02b Autoclaving 121°C20 min 16.69 ± 0.08b Extrusion 400 rpm 13.65 ± 0.50 b
c
Microwave 700 w 4 min 22.62 ± 0.07     Extrusion 500 rpm 7.50 ± 0.66a
Microwave 700 w 6 min 24.14 ± 0.36d        
Control 8.66 ± 0.02a Control 14.84 ± 0.21a Control 13.31 ± 0.16a
Steam heating 100°C 20 min 8.77 ± 0.07a Dry heating 105°C 30 min 18.71 ± 0.94b Infrared heating 105°C 30 min 14.08 ± 0.25a
Steam heating 100°C 40 min 8.65 ± 0.06a Dry heating 105°C 60 min 23.56 ± 0.27c Infrared heating 105°C 60 min 14.13 ± 0.21a
a d
Steam heating 100°C 60 min 8.42 ± 0.48 Dry heating 105°C 90 min 28.40 ± 0.16 Infrared heating 105°C 90 min 15.21 ± 0.20 b
Control 8.02 ± 0.00a Control 10.77 ± 0.26a Control 18.16 ± 0.01a
a a
4°C 72 hr 8.04 ± 0.17 Ultraviolet irradiation 6 hr 11.40 ± 0.27 Ultrasound 28 KHz 300 w 22.74 ± 0.72b
30 min
−18°C 72 hr 8.36 ± 0.25a Ultraviolet irradiation 11.21 ± 0.06a Ultrasound 28 KHz 300 w 25.47 ± 0.17c
12 hr 60 min
−80°C 72 hr 7.97 ± 0.01a Ultraviolet irradiation 10.70 ± 0.07a Ultrasound 28 KHz 300 w 26.98 ± 0.30 c
18 hr 90 min
Control 18.30 ± 0.36a Control 18.30 ± 0.36a    
a a
Pepsin 30 min 17.86 ± 0.22 Papain 30 min 18.70 ± 0.21    
Pepsin 60 min 17.98 ± 0.17a Papain 60 min 18.42 ± 0.16a    
a a
Pepsin 90 min 18.10 ± 0.24 Papain 90 min 18.56 ± 0.25    

Notes: Means with different superscript letter within the same column for each parameter are significantly different (p < .05).

total fatty acid in rice bran oil, the same as the Ozcan's research significant difference (p > .05) in the γ-oryzanol content by Amonrat
(Özcan, Al-Juhaimi, Ghafoor, Babiker, & Uslu, 2014). The results (Thanonkaew et al., 2012). There also have been some researches
showed that all the treatments had no significant influence (p > .05) about the loss of γ-oryzanol content after stabilization, for exam-
on the fatty acid composition. Similar findings were reported by ple, Shin indicated that a significant loss after extrusion at 130°C
Yılmaz, Tuncel, and Kocabıyık (2014) Maria (Irakli et al., 2018) and and 140°C (Shin et al., 2010). As for this phenomenon, Srisaipet and
Li et al. (2016) for infrared radiation heating. And Loypimai indi- Nuddagul put forward that the amount of γ-oryzanol did not vary
cated that both steaming and ohmic heating had no significant significantly (p > .05) with temperature lower than 120°C but a sig-
influence (p > .05) on the fatty acid composition of rice bran oil nificant change (p < .05) at higher than 120°C (Srisaipet & Nuddagul,
(Loypimai, Moongngarm, & Chottanom, 2015). Obviously, the fatty 2014). The temperature of stabilization methods in this research
acids were relatively stable under the different treatments. were all no higher than 120°C except autoclaving (121°C, only 1°C
higher than 120°C). This might prove that heating stabilization meth-
ods lower than 120°C had no significant influence on γ-oryzanol.
3.6 | Effects of different treatments on the
γ-oryzanol contents
3.7 | Effects of different treatments on the
The γ-oryzanol offers many health benefit for human, such as an- tocopherol contents
tioxidant, anticancer, and so on. Its antioxidant property was re-
ported higher than the four forms of vitamin E (Kurniawati, Yuliana, Rice bran oil is rich in tocopherols and tocotrienols (Khoei & Chekin,
& Budijanto, 2014). No significant changes of γ-oryzanol contents 2016). As shown in the Table 6, two tocopherols (α-tocotrienol and
were found in Table 6. Oryzanol is relatively stable to heat (Shin δ-tocopherol) were detected in the rice bran oil. But tocopherols
et al., 2010). Maria and Yilmaz reported that the γ-oryzanol content and tocotrienols are relatively unstable. A number of factors like
was not significant influenced (p > .05) by infrared radiation heating. heat, oxygen, alkali, light, and hydroperoxides can lead to the de-
Kanda indicated that γ-oryzanol was no significant difference by dif- composition of vitamin E (Zigoneanu, Williams, Xu, & Sabliov, 2008).
ferent heating methods and it was oxygen insensitive during heating From Table 6, it is easily found that all the treatments had no
(Rodchuajeen, Niamnuy, Charunuch, Soponronnarit, & Devahastin, significant influence (p > .05) on the δ-tocopherol contents. As for
2016). Hot air, roasting, steaming, and microwave were found no α-tocotrienol contents after most of the heating treatments such
YU et al. |
      11 of 14

TA B L E 5   Effects of different treatments on the fatty acid compositions

Treatment Palmitic acid Stearic acid Oleic acid Linoleic acid Arachidic acid Linolenic acid
a a a a a
Control 17.62 ± 0.05 2.17 ± 0.02 43.40 ± 0.07 34.75 ± 0.10 0.76 ± 0.01 1.40 ± 0.01a
a a a a a
Microwave 700 w 2 min 17.49 ± 0.26 2.16 ± 0.02 43.18 ± 0.43 34.99 ± 0.69 0.74 ± 0.04 1.44 ± 0.06a
Microwave 700 w 4 min 17.63 ± 0.08a 2.24 ± 0.03a 43.26 ± 0.17a 34.67 ± 0.10a 0.78 ± 0.04a 1.42 ± 0.00a
a a a a a
Microwave 700 w 6 min 17.66 ± 0.06 2.18 ± 0.00 43.45 ± 0.01 34.56 ± 0.04 0.78 ± 0.00 1.38 ± 0.01a
Control 17.62 ± 0.05a 2.17 ± 0.02a 43.40 ± 0.07a 34.75 ± 0.10a 0.76 ± 0.01a 1.40 ± 0.01a
a a a a a
Autoclaving 17.72 ± 0.04 2.16 ± 0.00 43.43 ± 0.04 34.54 ± 0.06 0.78 ± 0.00 1.36 ± 0.01a
121°C 20 min
Control 16.21 ± 0.10a 2.16 ± 0.02a 42.26 ± 0.07a 36.67 ± 0.02a 0.78 ± 0.01a 1.90 ± 0.01a
a a a a a
Extrusion 400 rpm 16.29 ± 0.05 2.15 ± 0.00 42.32 ± 0.13 36.64 ± 0.02 0.77 ± 0.02 1.89 ± 0.00a
Extrusion 500 rpm 16.31 ± 0.12a 2.17 ± 0.03a 42.28 ± 0.18a 36.58 ± 0.10a 0.78 ± 0.00a 1.90 ± 0.01a
a a a a a
Control 16.29 ± 0.02 2.15 ± 0.01 42.22 ± 0.07 36.67 ± 0.06 0.76 ± 0.00 1.90 ± 0.01a
Steam heating 100°C 20 min 16.51 ± 0.23a 2.20 ± 0.06a 42.09 ± 0.15a 36.54 ± 0.14a 0.77 ± 0.00a 1.88 ± 0.00a
a a a a a
Steam heating 100°C 40 min 16.42 ± 0.07 2.16 ± 0.00 42.22 ± 0.01 36.59 ± 0.02 0.77 ± 0.00 1.88 ± 0.01a
Steam heating 100°C 60 min 16.40 ± 0.10a 2.16 ± 0.01a 42.18 ± 0.04a 36.59 ± 0.02a 0.77 ± 0.00a 1.90 ± 0.03a
a a a a a
Control 16.08 ± 0.10 2.18 ± 0.03 42.39 ± 0.09 36.69 ± 0.02 0.78 ± 0.00 1.90 ± 0.01a
Dry heating 105°C 30 min 16.07 ± 0.11a 2.16 ± 0.00a 42.23 ± 0.08a 36.86 ± 0.18a 0.76 ± 0.01a 1.92 ± 0.01a
a a a a a
Dry heating 105°C 60 min 16.12 ± 0.01 2.13 ± 0.00 42.26 ± 0.01 36.82 ± 0.00 0.77 ± 0.00 1.90 ± 0.00a
Dry heating 105°C 90 min 16.32 ± 0.19a 2.16 ± 0.01a 42.22 ± 0.06a 36.62 ± 0.10a 0.77 ± 0.00a 1.89 ± 0.01a
a a a a a
Control 16.25 ± 0.02 2.19 ± 0.01 42.17 ± 0.02 36.73 ± 0.01 0.77 ± 0.00 1.94 ± 0.00a
Infrared heating 105°C 30 min 16.20 ± 0.12a 2.18 ± 0.02a 42.24 ± 0.02a 36.71 ± 0.14a 0.76 ± 0.00a 1.92 ± 0.00a
a a a a a
Infrared heating 105°C 60 min 16.18 ± 0.00 2.18 ± 0.01 42.20 ± 0.04 36.75 ± 0.07 0.78 ± 0.02 1.91 ± 0.00a
Infrared heating 105°C 90 min 16.33 ± 0.05a 2.17 ± 0.01a 42.16 ± 0.02a 36.79 ± 0.03a 0.76 ± 0.00a 1.93 ± 0.00a
a a a a a
Control 16.08 ± 0.10 2.18 ± 0.03 42.39 ± 0.09 36.69 ± 0.02 0.78 ± 0.00 1.90 ± 0.01a
4°C 72 hr 16.30 ± 0.01a 2.14 ± 0.00a 42.26 ± 0.01a 36.64 ± 0.02a 0.77 ± 0.00a 1.89 ± 0.00a
a a a a a
−18°C 72 hr 16.31 ± 0.12 2.16 ± 0.03 42.28 ± 0.18 36.58 ± 0.09 0.78 ± 0.00 1.90 ± 0.01a
−80°C 72 hr 16.25 ± 0.05a 2.15 ± 0.00a 42.23 ± 0.04a 36.69 ± 0.00a 0.77 ± 0.00a 1.90 ± 0.00a
a a a a a
Control 16.08 ± 0.08 2.19 ± 0.03 42.25 ± 0.12 36.76 ± 0.07 0.78 ± 0.00 1.93 ± 0.01a
Ultraviolet irradiation 6 hr 16.01 ± 0.08a 2.18 ± 0.01a 42.30 ± 0.04a 36.80 ± 0.13a 0.77 ± 0.01a 1.93 ± 0.01a
a a a a a
Ultraviolet irradiation 12 hr 16.04 ± 0.10 2.18 ± 0.01 42.26 ± 0.09 36.82 ± 0.02 0.78 ± 0.00 1.93 ± 0.00a
Ultraviolet irradiation 18 hr 16.01 ± 0.12a 2.18 ± 0.01a 42.39 ± 0.09a 36.72 ± 0.01a 0.78 ± 0.00a 1.92 ± 0.00a
a a a a a
Control 16.08 ± 0.08 2.19 ± 0.03 42.25 ± 0.12 36.76 ± 0.07 0.78 ± 0.00 1.93 ± 0.01a
Ultrasound 28 KHz 300 w 16.06 ± 0.05a 2.17 ± 0.00a 42.23 ± 0.03a 36.81 ± 0.06a 0.77 ± 0.00a 1.95 ± 0.02a
30 min
Ultrasound 28 KHz 300 w 16.05 ± 0.02a 2.17 ± 0.00a 42.22 ± 0.02a 36.83 ± 0.00a 0.77 ± 0.00a 1.96 ± 0.00a
60 min
Ultrasound 28 KHz 300 w 16.07 ± 0.07a 2.17 ± 0.00a 42.27 ± 0.03a 36.78 ± 0.04a 0.77 ± 0.00a 1.93 ± 0.01a
90 min
Control 15.93 ± 0.06a 2.18 ± 0.00a 42.32 ± 0.05a 36.72 ± 0.00a 0.78 ± 0.01a 2.07 ± 0.00a
Pepsin 30 min 15.90 ± 0.01a 2.17 ± 0.00a 42.36 ± 0.03a 36.70 ± 0.01a 0.78 ± 0.00a 2.06 ± 0.02a
a a a a a
Pepsin 60 min 15.96 ± 0.05 2.15 ± 0.01 42.30 ± 0.01 36.75 ± 0.03 0.77 ± 0.01 2.09 ± 0.01a
Pepsin 90 min 15.91 ± 0.08a 2.19 ± 0.01a 42.32 ± 0.07a 36.76 ± 0.02a 0.76 ± 0.02a 2.05 ± 0.01a
a a a a a
Control 15.93 ± 0.06 2.18 ± 0.00 42.32 ± 0.05 36.72 ± 0.00 0.78 ± 0.01 2.07 ± 0.00a
Papain 30 min 15.94 ± 0.03a 2.14 ± 0.03a 42.31 ± 0.02a 36.77 ± 0.03a 0.77 ± 0.01a 2.08 ± 0.01a
a a a a a
Papain 60 min 15.96 ± 0.01 2.17 ± 0.01 42.36 ± 0.02 36.70 ± 0.01 0.77 ± 0.00 2.09 ± 0.02a
Papain 90 min 15.97 ± 0.05a 2.19 ± 0.01a 42.30 ± 0.03a 36.74 ± 0.02a 0.76 ± 0.01a 2.05 ± 0.01a

Notes: Means with different superscript letter within the same column for each parameter are significantly different (p < .05).
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12 of 14      

TA B L E 6   Effect of different treatments on the γ-oryzanol and tocopherol contents of rice bran oil

Treatment γ-oryzanol (%) α-Tocotrienol (mg/g) δ-tocopherol (mg/g) Treatment γ-oryzanol (%) α-Tocotrienol (mg/g) δ-tocopherol (mg/g)
a a a a a
Control 0.28 ± 0.00 0.24 ± 0.03 0.12 ± 0.02 Control 0.28 ± 0.00 0.24 ± 0.03 0.12 ± 0.02a
Microwave 700 w 2 min 0.29 ± 0.01a 0.35 ± 0.02b 0.09 ± 0.00a Autoclaving 121°C 20 min 0.27 ± 0.02a 0.29 ± 0.03a 0.12 ± 0.02a
a a a
Microwave 700 w 4 min 0.31 ± 0.05 0.27 ± 0.00 0.09 ± 0.01        
Microwave 700 w 6 min 0.30 ± 0.00a 0.28 ± 0.03a 0.12 ± 0.02a        
a b a a ab
Control 0.33 ± 0.02 0.58 ± 0.01 0.14 ± 0.01 Control 0.36 ± 0.00 0.50 ± 0.03 0.23 ± 0.02ab
Extrusion 400 rpm 0.32 ± 0.05a 0.50 ± 0.02a 0.12 ± 0.02a Steam heating 100°C 20 min 0.38 ± 0.02a 0.46 ± 0.01a 0.15 ± 0.04a
a a a a bc
Extrusion 500 rpm 0.33 ± 0.01 0.51 ± 0.04 0.13 ± 0.01 Steam heating 100°C 40 min 0.37 ± 0.01 0.60 ± 0.04 0.17 ± 0.02a
        Steam heating 100°C 60 min 0.38 ± 0.01a 0.67 ± 0.00 c 0.34 ± 0.03b
a a a a a
Control 0.37 ± 0.00 0.31 ± 0.05 0.14 ± 0.01 Control 0.38 ± 0.04 0.42 ± 0.01 0.14 ± 0.02a
Dry heating 105°C 30 min 0.37 ± 0.01a 0.40 ± 0.02b 0.25 ± 0.09a Infrared heating 105°C 30 0.39 ± 0.00a 0.86 ± 0.13c 0.19 ± 0.08a
min
Dry heating 105°C 60 min 0.38 ± 0.01a 0.41 ± 0.03b 0.22 ± 0.01a Infrared heating 105°C 60 0.39 ± 0.01a 0.74 ± 0.02c 0.19 ± 0.03a
min
Dry heating 105°C 90 min 0.38 ± 0.01a 0.32 ± 0.10a 0.19 ± 0.02a Infrared heating 105°C 90 0.37 ± 0.03a 0.67 ± 0.03b 0.16 ± 0.03a
min
Control 0.37 ± 0.00a 0.31 ± 0.05a 0.14 ± 0.01a Control 0.31 ± 0.02a 0.32 ± 0.03a 0.15 ± 0.02a
4°C 72 hr 0.36 ± 0.01a 0.31 ± 0.08a 0.13 ± 0.02a Ultraviolet irradiation 6 hr 0.31 ± 0.05a 0.28 ± 0.06a 0.16 ± 0.02a
a a a a a
−18°C 72 hr 0.34 ± 0.03 0.34 ± 0.07 0.18 ± 0.01 Ultraviolet irradiation 12 hr 0.31 ± 0.05 0.37 ± 0.15 0.12 ± 0.02a
−80°C 72 hr 0.34 ± 0.00a 0.32 ± 0.06a 0.12 ± 0.03a Ultraviolet irradiation 18 hr 0.32 ± 0.08a 0.32 ± 0.03a 0.12 ± 0.01a
a a a a a
Control 0.31 ± 0.02 0.32 ± 0.03 0.15 ± 0.02 Control 0.34 ± 0.00 0.69 ± 0.01 0.14 ± 0.01a
Ultrasound 28 KHz 300 w 30 min 0.34 ± 0.01a 0.55 ± 0.03b 0.12 ± 0.02a Pepsin 30 min 0.34 ± 0.01a 0.65 ± 0.02a 0.14 ± 0.02a
a ab a a a
Ultrasound 28 KHz 300 w 60 min 0.30 ± 0.04 0.48 ± 0.04 0.12 ± 0.06 Pepsin 60 min 0.33 ± 0.01 0.70 ± 0.01 0.13 ± 0.01a
Ultrasound 28 KHz 300 w 90 min 0.32 ± 0.01a 0.71 ± 0.09b 0.16 ± 0.00a Pepsin 90 min 0.35 ± 0.02a 0.68 ± 0.01a 0.11 ± 0.02a
a a a
Control 0.34 ± 0.00 0.69 ± 0.01 0.14 ± 0.01        
Papain 30 min 0.33 ± 0.02a 0.67 ± 0.02a 0.14 ± 0.02a        
a a a
Papain 60 min 0.35 ± 0.01 0.66 ± 0.03 0.16 ± 0.02        
Papain 90 min 0.34 ± 0.00a 0.68 ± 0.02a 0.13 ± 0.01a        

Notes: Means with different superscript letter within the same column for each parameter are significantly different (p < .05).
YU et al.
YU et al. |
      13 of 14

as microwave, steam heating, autoclaving, dry heating, and infrared C O N FL I C T O F I N T E R E S T

heating, significant increases (p < .05) were easily found. Moreau The authors have declared no conflicts of interest for this article.
indicated that the extractability of tocopherols in corn hulls could
be increased after heat pretreatment, suggesting that a significant ORCID
amount of tocopherol was bound to proteins, phosphate, or phos- Cheng-wei Yu  https://orcid.org/0000-0001-5337-9200
pholipid, and heating treatment could break the bonds (Moreau, Qi-rui Hu  https://orcid.org/0000-0003-4842-4147
And, & Powell, 1999). Zigoneanu found that the content of vitamin Ze-yuan Deng  https://orcid.org/0000-0001-5650-1507
E increased with the temperature increasing from 40°C to 120°C
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